The joining of DNA fragments is central to the methodology of molecular biology. There is a consistent need to efficiently join DNA segments to form useful, informative arrays that can allow for analysis in vitro and in vivo. DNA restriction endonucleases, in combination with DNA ligase, have been the principal tools used to create such fragment arrays. This approach relies heavily upon the natural occurrence of appropriate restriction endonuclease recognition sites, and to a lesser extent on being able to insert appropriate sites through such techniques as site-directed mutagenesis, PCR or linker insertion.
The primary method of joining DNA fragments involves enzymatic ligation, preferably with cohesive termini created by restriction endonuclease cleavage such that the two fragments can only be joined in a single orientation. Often, the product of that ligation is a circular molecule suitable for transformation into and propagation in a bacterial host. Alternatively, the cohesive termini may be identical, in which case two possible orientations can result, necessitating screening of the final products. Finally, one or both of the termini may be blunt ends, reducing the efficiency of ligase joining, but also eliminating the requirement for compatible cohesive termini. In a practical sense, the number of elements that can be joined is limited to two, possibly three elements. In addition, in the absence of DNA ligase, no joining is observed.
One naturally-occurring method that increases the efficiency of fragment joining is observed in the life cycle of the bacteriophage lambda. Upon lambda infection, a linear double-stranded genome enters the cell. This genome is circularized prior to replication via pairing of complementary 12-nucleotide single-stranded regions at the two ends of the genome. These single-stranded regions are created after replication and prior to phage packaging by the action of the lambda int gene product. The int gene product can be used in vitro in much the same way as restriction endonucleases to cut and rejoin DNA fragments. In theory, this approach could be expanded to join multiple elements in a defined, ordered array. However, this would require multiple int-like proteins that recognize different sequences to assure unique orientation of fragments. This could be accomplished by using similar regions from different bacteriophages (e.g., bacteriophage P22), but this would require the isolation of a separate int gene product for each set of cohesive ends, and potentially introduce difficulties in optimizing reaction conditions for more than one cleavage. Additionally, in this approach the cohesive sequence is constrained to the naturally occurring sequence. Thus, while this is a possible approach, it is not the optimal approach.
An alternative is to create single-stranded regions by the combined action of nucleases. Several methodologies have been described, including: limiting digestion by controlling the time of digestion (Li and Evans Nucleic Acids Res. 15:4165-4166 (1997)), inhibiting digestion at a selected location (Aslanidis and de Jong Nucleic Acids Res. 18:6069-6074 (1990); Zhou and Hatahet Nucleic Acids Res. 23:1089-1090 (1995); Dietmaier, et al. Nucleic Acids Res. 21:3603-3604 (1993)) and selectively enhancing digestion at a specific location (U.S. Pat. No. 5,137,814; Nisson et al. PCR Methods Appl. 1:20-123 (1991)). In still another method, a mixture of staggered PCR products are hybridized together to create overhangs (Tillett and Nelian Nucleic Acids Res. 27: e26 (1999)). Most of these methods require a PCR step to add a DNA sequence element or non-standard nucleotide to the termini as a prelude to exonuclease action. The inherent infidelity of PCR raises concerns of introducing a mutation into the amplified DNA sequence, thus it would be more desirable to assemble DNA fragments replicated in vivo. Furthermore, it is difficult to assess whether the desired enzymatic action has been completed on the DNA termini since the gross properties of the fragment (e.g., electrophoretic mobility) are largely unaltered.
The present invention is related to the production of defined single-stranded regions in DNA, and the use of such regions to join, detect and purify such molecules. Site-specific DNA nicking endonucleases are used to form the single-stranded regions by nicking at the boundaries of the single-strand regions, either on opposing DNA strands (creating terminal single-stranded regions) or on the same strand (creating a single-strand gap).
Creation of such single-stranded regions facilitates assembly of multiple nucleic acid fragments in an ordered array, either linear or circular. This is useful in a variety of applications, including construction of vectors with interchangeable cassettes. Although the examples provided here use the enzyme N.BstNBI, the skilled artisan will appreciate that any other site-specific nicking enzyme would give equivalent results.